A large body of evidence indicates that cancer has a genetic basis, and that tumor progression is a multi-step, complicated set of events. The analysis of the transforming oncogenes has contributed significantly to our understanding of neoplasia, but many of the crucial causative steps remain elusive. Progress is hampered by our poor understanding of how proliferation signals are transmitted form the cell surface to the nucleus, and how they are executed. Two basic questions are of great importance. What are the normal functions of the cellular proto-oncogenes in cellular physiology? What are the links in the chain of command downstream from the oncogenes? Three proto-oncogenes, c-src, c-raf, and c-myc were chosen for the present study because each appears to act at a distinct and different point in the signal transduction process. The primary aim of this proposal is the elucidation of the role of these proto-oncogenes in normal cellular physiology. This goal will be approached using targeted gene modification. Since many biological phenomena can be profitably approached in tissue culture, all experiments will be conducted in appropriate in vitro model systems. The aim will be to use the phenotypes of the resultant null mutants as clues to unravel the molecular functions of the genes in question. In addition, it is hoped that gene targeting will provide an entry point for future genetic studies of the proto-oncogenes. Progress in the field of gene targeting in mammalian cells has been very rapid during 1988. Four distinct genetic selection methods for the recovery of homologous recombination events at nonselected chromosomal loci have been independently developed (Doetschman et al., 1988; Jasin and Berg, 1988; Mansour et al., 1988; Sedivy and Sharp, 1989). Gene targeting in cultured cells necessitates the modification of multiple gene copies in diploid or even polyploid backgrounds. This will be accomplished by performing targeted homologous recombination several times in succession. To circumvent the possible lethality of homozygous null phenotypes, a conditionally expressed copy of the target gene will be introduced into the genome. This will allow the propagation of a cell line in which both endogenous copies of the target gene are modified. Then, under conditions non-permissive for expression of the introduced gene copy, the null phenotype will become apparent and will be subjected to experimental analysis. Another aim of this proposal is to compare, generalize, and further develop the methods of gene targeting.